Small molecules that replace or agonize p53 function

ABSTRACT

This invention provides a novel screening system for identifying p53 mimetics/agonists. Also provided are small organic molecules that act as effective p53 mimetics/agonists.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims benefit of and priority to U.S. Ser. No.60/603,308, filed Aug. 20, 2004, which is incorporated herein byreference in its entirety for all purposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[NOT APPLICABLE]

FIELD OF THE INVENTION

This invention pertains to the field of oncology. In particular thisintention provides compounds that replace and/or agonize p53 functionand methods of identifying such compounds.

BACKGROUND OF THE INVENTION

Two-thirds of all breast cancers display mutations in the tumorsuppressor p53 (Lai et al. (2004) Breast Cancer Res. Treat., 83: 57-66),and one-third of all advanced human breast carcinomas demonstrate amarked reduction in expression of the pro-apoptotic Bcl-2 family memberBax (Krajewski et al. (1995) Cancer Res., 55: 4471-4478). Bax and p53mutations are associated with a high percentage of all human tumors. Thesubgroup of patients displaying p53 mutations or reduced Bax expressiongenerally respond poorly to therapy and exhibit rapidly growing tumorsand shorter median survival (Lai et al., supra; Reed (1996) J. Clin.Invest., 97:2403-2404). Though extensively pursued, the mechanism bywhich Bax expression is regulated in normal, malignant, or dying cellsis unknown. The only known endogenous activator of Bax expression is thep53 transcription factor, which is responsible for inducing cell deathin cancerous and damaged cells (Miyashita and Reed (1995) Cell, 80:293-299).

SUMMARY OF THE INVENTION

This invention provides novel screening systems well suited foridentification of agents that act as p53 mimetics/agonists. Alsoprovided are effective p53 mimetics/agonists.

Thus, in certain embodiments, this invention provides compound thatpromotes cell death in a p53 naive cell (e.g. a tumor cell). Certainpreferred compounds are those of Formula I herein, where R¹ and R⁴ areindependently selected from the group consisting of an amino group, acyano group, a nitro group, a carboxyl group, halo, hydroxyl, SO₂, C₁₋₆alkyl, C₁₋₆ haloalkyl, C₁₋₈ alkoxyl, C₁₋₁₁ alkoxyalkyl, C₁₋₆ alkylamino,C₁₋₆ aminoalkyl, and the like; R² and R³ are independently selected fromthe group consisting of C, N, and the like; R⁵ is C or O; R⁶ and R⁷ areindependently selected from the group consisting of H, H, halogen, CN,NO₂, C₁₋₁₀ branched or unbranched saturated or unsaturated alkyl, C₁₋₁₀branched or unbranched alkoxy, C₁₋₁₀ branched or unbranched acyl, C₁₋₁₀branched or unbranched acyloxy, C₁₋₁₀ branched or unbranched alkylthio,aminosulfonyl, aryl, aroyl, aryloxy, arylsulfonyl, heteroarylheteroaryloxy, and the like; R⁸ is selected from the group consisting ofNO₂, OH, COOH, and the like; R⁹ is selected from the group consisting ofH, CH₃, and the like, or a pharmaceutically acceptable ester or saltthereof. In certain embodiments R⁶ and R⁷ are both halogen. In variousembodiments R¹ and R⁴ are both amino. In various embodiments R² and R³are both N. In various embodiments R⁸ is NO₂. In certain embodiments thecompound has the formula of formula II.

Also provided is a method of promoting cell death in a p53 naive cell(e.g. a cancer cell including solid tumors, metastatic cells andnon-solid tumor cancers). The method typically involves contacting thecell with a compound that induces transcription of ras. In variousembodiments the compound is a compound according to Formula I asdescribed herein. In various embodiments the compound is in apharmaceutically acceptable excipient. In certain embodiments the cellis a liver cancer cell, a breast cancer cell, a colon cancer cell, alung cancer cell, a uterine cancer cell, an ovarian cancer cell, aprostate cancer cell, or a colon cancer cell.

Also provided is a cell line for screening for agents that promotes celldeath in a p53 naive cell. The cell line typically comprises mammaliancells containing a nucleic acid construct comprising bax promoterelements comprising a 5′ UTR, a TATAA sequence, and one or moreconsensus sequences that bind p53, where the bax promoter elements areoperably linked to a reporter such that binding of a p53 protein to oneor more of said consensus sequences induces or increases transcriptionof said reporter. In certain embodiments the reporter is a fluorescentprotein, luciferase, chloramphenicol acetyl transferase (CAT),β-galactosidase (β-gal), alkaline phosphatase, horse radish peroxidase(HRP), or a growth hormone (GH). In certain embodiments the reportercomprises a luciferin gene or cDNA. In various embodiments the mammaliancell is a p53 naïve cell (e.g., a HEK293T cell).

This invention also provides methods of identifying agents that promotecell death in p53 naive cells. The methods typically involve contactingone or more cells from the cell line described herein with one or moretest agents; detecting a signal from the reporter in the cell wherein anincrease in signal from the reporter indicates that the agent is anagent that is likely to promote cell death in a p53 naive cell. Incertain embodiments a plurality of test agents are contacted to saidcell at the same time. In certain embodiments a single test agent iscontacted to said cell. In various embodiments the increase is measuredrelative to a control lacking the test agent or comprising the testagent at a lower concentration. In certain embodiments the method isperformed by a robotic system. In various embodiments the test agentsare from a library selected from the group consisting of Chem BridgeDiverSet E, Bionet 1, CEREP, Maybridge 1, Maybridge 2, Peakdale 1,Peakdale 2, ChemDiv Combilab and International, Mixed Commercial Plate1, Mixed Commercial Plate 2, Mixed Commercial Plate 3, Mixed CommercialPlate 4, ChemBridge Microformat, Commercial Diversity Set1, NCIStructural Diversity Set version 1, NCI Structural Diversity Set version2, NCI Mechanistic Diversity Set, NCI Open Collection 1, NCI OpenCollection 2, NINDS Custom Collection, SpecPlus Collection, BIOMOL ICCBKnown Bioactives, ICCB Discretes Collections, ICCB2, ICCB3, ICCB4, NCIMarine Extracts, Aqueous fractions—NCI Plant and Fungal Extracts,Organic fractions—NCI Plant and Fungal Extracts, Philippines PlantExtracts 1, Philippines Plant Extracts 2, and Starr Foundation Extracts1.

DEFINITIONS

A “p53 naive cell” refers to a cell that expresses a defective p53,and/or a p53 with reduced activity, and/or that under expresses normalp53, etc. Typically p53 naive cells show reduced p53 tumor suppressoractivity as compared to a “normal” cell.

The terms “reporter” or “reporter gene” refer to gene or cDNA thatexpresses a product that is detectable by spectroscopic, photochemical,biochemical, immunochemical, electrical, optical, and/or chemical means.Useful reporters/labels in this regard include, but are not limited toluminescent proteins, fluorescent proteins (e.g. green fluorescentprotein (GFP), red fluorescent protein (RFP), etc.), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase, β-galactosidase, andothers commonly used in an ELISA), and the like.

The term “reporter gene operably linked to a promoter” refers to apromoter and a reporter gene disposed such that the promoter regulatestranscription of the reporter gene.

The term “test agent” refers to an agent that is to be screened in oneor more of the assays described herein. The agent can be virtually anychemical compound. It can exist as a single isolated compound or can bea member of a chemical (e.g. combinatorial) library. In a particularlypreferred embodiment, the test agent will be a small organic molecule.

The term “small organic molecule” refers to a molecule of a sizecomparable to those organic molecules generally used in pharmaceuticals.The term excludes biological macromolecules (e.g., proteins, nucleicacids, etc.). Preferred small organic molecules range in size up toabout 5000 Da, more preferably up to 2000 Da, and most preferably up toabout 1000 Da.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates p53 binding elements in the Bax 5′ UTR.

FIG. 2A shows the structure of Rp53-1 (G6). FIG. 2B shows that Rp53-1(G6) activates the p53/Bax reporter in HEK293T cells to levels relativeto transiently overexpressed p53.

FIG. 3 shows that p53-mutant (MDA-435), but not wild-type (MCF-7),breast cancer cells are sensitive to the p53-mimetic compound Rp53-1(G6).

FIG. 4 illustrates p53 mutations that result in dysregulation ofBax/Bcl-2 expression.

FIG. 5 illustrates that bax expression is induced by p53 and leads toapoptosis.

FIG. 6 illustrates screening for p53 mimetics/agonists.

FIG. 7 shows that a single pulse treatment of breast cancer cells withG6 results in complete drug efficacy.

FIG. 8 shows that G6 induces p53 Reporter in p53 Mt and Null Cells.

FIG. 9 illustrates the results of treatment of HT-29 cells with G6.

FIG. 10 shows that G6 treatment of nude mice implanted with humanosteosarcoma tissue results in complete regression of tumor.

DETAILED DESCRIPTION

This invention is based in part, on the idea that screening for smallmolecular compounds that act as p53 mimics in activating bax expressioncan yield therapeutics for many mammary neoplasias.

I. Screening System for p53 Mimetics/Agonists

To identify p53 mimics, we developed a live mammalian cell system thatreports the promotion of the expression of the pro-apoptotic Baxprotein. The bax promoter consists of a 372 nucleotide 5′ UTR, a TATAAsequence, and several consensus sequences that bind p53, resulting inup-regulation of Bax expression (see, e.g., FIG. 1). By expressing thesesites upstream of the gene encoding luciferase in HEK293T cells (see,e.g., FIG. 5), we have established a novel set of reporter cell lines.These cells provide a system which allows identification of both directactivators of Bax expression and replacements of an apoptotic functionof p53 (see, e.g., FIG. 6).

A) High throughput Screening for p53 Mimetics/Agonists.

The cell lines described herein are particularly effective for screeningfor p53 mimetics/agonists. In addition, these cell lines are also wellsuited to “high-throughput” modalities. Conventionally, new chemicalentities with useful properties (e.g., modulation of transporteractivity or expression, or ability to be transported by the transportersof this invention) are generated by identifying a chemical compound(called a “lead compound”) with some desirable property or activity,creating variants of the lead compound, and evaluating the property andactivity of those variant compounds. However, the current trend is toshorten the time scale for all aspects of drug discovery. Because of theability to test large numbers quickly and efficiently, high throughputscreening (HTS) methods are replacing conventional lead compoundidentification methods.

In one preferred embodiment, high throughput screening methods involveproviding a library containing a large number of compounds (candidatecompounds/test agents) potentially having the desired activity. Such“chemical libraries” are then screened in one or more assays, asdescribed herein, to identify those library members (particular chemicalspecies or subclasses) that display a desired characteristic activity.The compounds thus identified can serve as conventional “lead compounds”or can themselves be used as potential or actual therapeutics.

1) Libraries for Screening for Agents that Act as p53 Mimetics/Agonists.

The likelihood of an assay identifying an agent that acts as a p53mimetic/agonist is increased when the number and types of test agentsused in the screening system is increased. Recently, attention hasfocused on the use of combinatorial chemical libraries to assist in thegeneration of new chemical compound leads. A combinatorial chemicallibrary is a collection of diverse chemical compounds generated byeither chemical synthesis or biological synthesis by combining a numberof chemical “building blocks” such as reagents. For example, a linearcombinatorial chemical library such as a polypeptide library is formedby combining a set of chemical building blocks called amino acids inevery possible way for a given compound length (i.e., the number ofamino acids in a polypeptide compound). Millions of chemical compoundscan be synthesized through such combinatorial mixing of chemicalbuilding blocks. For example, one commentator has observed that thesystematic, combinatorial mixing of 100 interchangeable chemicalbuilding blocks results in the theoretical synthesis of 100 milliontetrameric compounds or 10 billion pentameric compounds (Gallop et al.(1994) 37(9): 1233-1250).

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka (1991) Int. J. Pept. Prot. Res., 37:487-493, Houghton et al. (1991) Nature, 354: 84-88). Peptide synthesisis by no means the only approach envisioned and intended for use withthe present invention. Other chemistries for generating chemicaldiversity libraries can also be used. Such chemistries include, but arenot limited to: peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991),encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), randombio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992),benzodiazepines (U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., (1993) Proc.Nat. Acad. Sci. USA 90: 6909-6913), vinylogous polypeptides (Hagihara etal. (1992) J. Amer. Chem. Soc. 114: 6568), nonpeptidal peptidomimeticswith a Beta-D-Glucose scaffolding (Hirschmann et al., (1992) J. Amer.Chem. Soc. 114: 9217-9218), analogous organic syntheses of smallcompound libraries (Chen et al. (1994) J. Amer. Chem. Soc. 116: 2661),oligocarbamates (Cho, et al., (1993) Science 261:1303), and/or peptidylphosphonates (Campbell et al., (1994) J. Org. Chem. 59: 658). See,generally, Gordon et al., (1994) J. Med. Chem. 37:1385, nucleic acidlibraries (see, e.g., Strategene, Corp.), peptide nucleic acid libraries(see, e.g., U.S. Pat. No. 5,539,083) antibody libraries (see, e.g.,Vaughn et al. (1996) Nature Biotechnology, 14(3): 309-314), andPCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al. (1996)Science, 274: 1520-1522, and U.S. Pat. No. 5,593,853), and small organicmolecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, Jan18, page 33, isoprenoids U.S. Pat. No. 5,569,588, thiazolidinones andmetathiazanones U.S. Pat. No. 5,549,974, pyrrolidines U.S. Pat. Nos.5,525,735 and 5,519,134, morpholino compounds U.S. Pat. No. 5,506,337,benzodiazepines U.S. Pat. No. 5,288,514, and the like).

In addition, a number of libraries are commercially available. Suchlibraries include, but are not limited to the Chem Bridge DiverSet E(16,320 compounds), Bionet 1 (4,800 compounds), CEREP (4,800compounds), Maybridge 1 (8,800 compounds), Maybridge 2 (704 compounds),Peakdale 1 (2,816 compounds), Peakdale 2 (352 compounds), ChemDivCombilab and International (28,864 compounds), Mixed Commercial Plate 1(352 compounds), Mixed Commercial Plate 2 (320 compounds), MixedCommercial Plate 3 (251 compounds), Mixed Commercial Plate 4 (331compounds), ChemBridge Microformat (50,000 compounds), CommercialDiversity Set1 (5,056 compounds), various NCI collections (e.g.Structural Diversity Set, version 1 (1,900 compounds), StructuralDiversity Set, version 2 (1,900 compounds), Mechanistic Diversity Set(879 compounds), Open Collection 1 (90,000 compounds), Open Collection 2(10,240 compounds), and the like, NINDS Custom Collection (1,040compounds), ICCB Bioactives 1 (489 compounds), SpecPlus Collection (960compounds), BIOMOL ICCB Known Bioactives (480 compounds), various ICCBDiscretes Collections (e.g., ICCB1 (190 compounds), ICCB2 (352compounds), ICCB3 (352 compounds), ICCB4 (352 compounds), and the like),various natural product extracts (e.g., NCI Marine Extracts (352 wells),Aqueous fractions—NCI Plant and Fungal Extracts (2,112 wells), Organicfractions—NCI Plant and Fungal Extracts (1,408 wells), Philippines PlantExtracts 1 (200 wells), Philippines Plant Extracts 2 (648 wells), StarrFoundation Extracts 1 (1024 wells)) and the like.

2) High Throughput Screening Devices.

A number of high throughput screening systems are commercially available(see, e.g., Zymark Corp., Hopkinton, Mass.; Air Technical Industries,Mentor, Ohio; Beckman Instruments, Inc. Fullerton, Calif.; PrecisionSystems, Inc., Natick, Mass., etc.). These systems typically automateentire procedures including all sample and reagent pipetting, liquiddispensing, timed incubations, and final readings of the microplate indetector(s) appropriate for the assay. These configurable systemsprovide high throughput and rapid start up as well as a high degree offlexibility and customization. The manufacturers of such systems providedetailed protocols the various high throughput. Thus, for example,Zymark Corp. provides technical bulletins describing screening systemsfor detecting the modulation of gene transcription, ligand binding, andthe like.

II. P53 Mimetics/Agonists.

In certain embodiments, this invention provides a number of p53mimetics/agonists. These compound typically promotes cell death in a p53naive cell (e.g. a cancer cell). In certain embodiments, the compoundscomprising the formula:

where R¹ and R⁴ are independently selected from the group consisting ofan amino group, a cyano group, a nitro group, a carboxyl group, halo,hydroxyl, SO₂, C₁₋₆ alkyl, C₁₋₆ haloalkyl, C₁₋₈ alkoxyl, C₁₋₁₁alkoxyalkyl, C₁₋₆ alkylamino, and C₁₋₆ aminoalkyl; R² and R³ areindependently selected from the group consisting of C, N; R⁵ is C or O;R⁶ and R⁷ are independently selected from the group consisting of H, H,halogen, CN, NO₂, C₁₋₁₀ branched or unbranched saturated or unsaturatedalkyl, C₁₋₁₀ branched or unbranched alkoxy, C₁₋₁₀ branched or unbranchedacyl, C₁₋₁₀ branched or unbranched acyloxy, C₁₋₁₀ branched or unbranchedalkylthio, aminosulfonyl, aryl, aroyl, aryloxy, arylsulfonyl, heteroaryland heteroaryloxy; R⁸ is selected from the group consisting of NO₂, OH,COOH; R⁹ is selected from the group consisting of H, CH₃. Also includedare or a pharmaceutically acceptable esters or salts of such compounds.In certain embodiments, R⁶ and R⁷ are both halogen. In certainembodiments, R′ and R⁴ are both amino. In certain embodiments, R³ and R³are both N. In certain embodiments, R⁸ is NO₂. In certain embodiments,the compound has the formula:

Such compounds can readily be formulated, e.g. using the compound offormula II as a starting point.

III. Pharmaceutical Formulations.

In certain embodiments, the p53 mimetics/agonists of this invention areprovided and/or administered as pharmaceutical formulations. Typicallyone or more p53 mimetics/agonists of this invention are administered,e.g. to an individual diagnosed as having one or more symptoms ofcancer. The p53 mimetics/agonists can be administered in the “native”form or, if desired, in the form of salts, esters, amides, prodrugs,derivatives, and the like, provided the salt, ester, amide, prodrug orderivative is suitable pharmacologically, i.e., effective in the presentmethod. Salts, esters, amides, prodrugs and other derivatives of theactive agents may be prepared using standard procedures known to thoseskilled in the art of synthetic organic chemistry and described, forexample, by March (1992) Advanced Organic Chemistry; Reactions,Mechanisms and Structure, 4th Ed. N.Y. Wiley-Interscience.

For example, acid addition salts are prepared from the free base usingconventional methodology that typically involves reaction with asuitable acid. Generally, the base form of the drug is dissolved in apolar organic solvent such as methanol or ethanol and the acid is addedthereto. The resulting salt either precipitates or may be brought out ofsolution by addition of a less polar solvent. Suitable acids forpreparing acid addition salts include both organic acids, e.g., aceticacid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, malicacid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid, and the like, as well as inorganic acids, e.g.,hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like. An acid addition salt may be reconvertedto the free base by treatment with a suitable base. Particularlypreferred acid addition salts of the active agents herein are halidesalts, such as may be prepared using hydrochloric or hydrobromic acids.Conversely, preparation of basic salts of the p53 mimetics/agonists areprepared in a similar manner using a pharmaceutically acceptable basesuch as sodium hydroxide, potassium hydroxide, ammonium hydroxide,calcium hydroxide, trimethylamine, or the like. Particularly preferredbasic salts include alkali metal salts, e.g., the sodium salt, andcopper salts.

Preparation of esters typically involves functionalization of hydroxyland/or carboxyl groups which may be present within the molecularstructure of the drug. The esters are typically acyl-substitutedderivatives of free alcohol groups, i.e., moieties that are derived fromcarboxylic acids of the formula RCOOH where R is alky, and preferably islower alkyl. Esters can be reconverted to the free acids, if desired, byusing conventional hydrogenolysis or hydrolysis procedures.

Amides and prodrugs may also be prepared using techniques known to thoseskilled in the art or described in the pertinent literature. Forexample, amides may be prepared from esters, using suitable aminereactants, or they may be prepared from an anhydride or an acid chlorideby reaction with ammonia or a lower alkyl amine. Prodrugs are typicallyprepared by covalent attachment of a moiety that results in a compoundthat is therapeutically inactive until modified by an individual'smetabolic system.

The mimetics/agonists identified herein are useful for parenteral,topical, oral, nasal (or otherwise inhaled), rectal, or localadministration, such as by aerosol or transdermally, for prophylacticand/or therapeutic treatment of atherosclerosis and/or symptoms thereof.The pharmaceutical compositions can be administered in a variety of unitdosage forms depending upon the method of administration. Suitable unitdosage forms, include, but are not limited to powders, tablets, pills,capsules, lozenges, suppositories, patches, nasal sprays, injectibles,implantable sustained-release formulations, lipid complexes, etc.

The mimetics/agonists of this invention are typically combined with apharmaceutically acceptable carrier (excipient) to form apharmacological composition. Pharmaceutically acceptable carriers cancontain one or more physiologically acceptable compound(s) that act, forexample, to stabilize the composition or to increase or decrease theabsorption of the active agent(s). Physiologically acceptable compoundscan include, for example, carbohydrates, such as glucose, sucrose, ordextrans, antioxidants, such as ascorbic acid or glutathione, chelatingagents, low molecular weight proteins, protection and uptake enhancerssuch as lipids, compositions that reduce the clearance or hydrolysis ofthe active agents, or excipients or other stabilizers and/or buffers.

Other physiologically acceptable compounds include wetting agents,emulsifying agents, dispersing agents or preservatives that areparticularly useful for preventing the growth or action ofmicroorganisms. Various preservatives are well known and include, forexample, phenol and ascorbic acid. One skilled in the art wouldappreciate that the choice of pharmaceutically acceptable carrier(s),including a physiologically acceptable compound depends, for example, onthe route of administration of the active agent(s) and on the particularphysio-chemical characteristics of the active agent(s).

The excipients are preferably sterile and generally free of undesirablematter. These compositions may be sterilized by conventional, well-knownsterilization techniques.

In therapeutic applications, the compositions of this invention areadministered to a patient suffering from one or more symptoms ofatherosclerosis or at risk for atherosclerosis in an amount sufficientto cure or at least partially prevent or arrest the disease and/or itscomplications. An amount adequate to accomplish this is defined as a“therapeutically effective dose.” Amounts effective for this use willdepend upon the severity of the disease and the general state of thepatient's health. Single or multiple administrations of the compositionsmay be administered depending on the dosage and frequency as requiredand tolerated by the patient. In any event, the composition shouldprovide a sufficient quantity of the active agents of the formulationsof this invention to effectively treat (ameliorate one or more symptoms)the patient.

The concentration of p53 mimetics/agonists can vary widely, and will beselected primarily based on fluid volumes, viscosities, body weight andthe like in accordance with the particular mode of administrationselected and the patient's needs. Concentrations, however, willtypically be selected to provide dosages ranging from about 0.1 or 1mg/kg/day to about 50 mg/kg/day and sometimes higher. Typical dosagesrange from about 3 mg/kg/day to about 3.5 mg/kg/day, preferably fromabout 3.5 mg/kg/day to about 7.2 mg/kg/day, more preferably from about7.2 mg/kg/day to about 11.0 mg/kg/day, and most preferably from about11.0 mg/kg/day to about 15.0 mg/kg/day. In certain preferredembodiments, dosages range from about 10 mg/kg/day to about 50mg/kg/day. It will be appreciated that such dosages may be varied tooptimize a therapeutic regimen in a particular subject or group ofsubjects.

In certain preferred embodiments, the p53 mimetics/agonists of thisinvention are administered orally (e.g. via a tablet) or as aninjectable in accordance with standard methods well known to those ofskill in the art. In other preferred embodiments, the p53mimetics/agonists, may also be delivered through the skin usingconventional transdermal drug delivery systems, i.e., transdermal“patches” wherein the active agent(s) are typically contained within alaminated structure that serves as a drug delivery device to be affixedto the skin. In such a structure, the drug composition is typicallycontained in a layer, or “reservoir,” underlying an upper backing layer.It will be appreciated that the term “reservoir” in this context refersto a quantity of “active ingredient(s)” that is ultimately available fordelivery to the surface of the skin. Thus, for example, the “reservoir”may include the active ingredient(s) in an adhesive on a backing layerof the patch, or in any of a variety of different matrix formulationsknown to those of skill in the art. The patch may contain a singlereservoir, or it may contain multiple reservoirs.

In one embodiment, the reservoir comprises a polymeric matrix of apharmaceutically acceptable contact adhesive material that serves toaffix the system to the skin during drug delivery. Examples of suitableskin contact adhesive materials include, but are not limited to,polyethylenes, polysiloxanes, polyisobutylenes, polyacrylates,polyurethanes, and the like. Alternatively, the drug-containingreservoir and skin contact adhesive are present as separate and distinctlayers, with the adhesive underlying the reservoir which, in this case,may be either a polymeric matrix as described above, or it may be aliquid or hydrogel reservoir, or may take some other form. The backinglayer in these laminates, which serves as the upper surface of thedevice, preferably functions as a primary structural element of the“patch” and provides the device with much of its flexibility. Thematerial selected for the backing layer is preferably substantiallyimpermeable to the active agent(s) and any other materials that arepresent.

In certain instances, particularly where bioavailability is low withsystemic administration, the p53 mimetics/agonists are administereddirectly to a tumor site or to a post-operative tumor site. Suchdelivery may be direct through a cannula or by injection or placement ofa time-release formulation, e.g. during a surgical procedure.

Other preferred formulations for topical drug delivery include, but arenot limited to, ointments and creams. Ointments are semisolidpreparations which are typically based on petrolatum or other petroleumderivatives. Creams containing the selected active agent, are typicallyviscous liquid or semisolid emulsions, often either oil-in-water orwater-in-oil. Cream bases are typically water-washable, and contain anoil phase, an emulsifier and an aqueous phase. The oil phase, alsosometimes called the “internal” phase, is generally comprised ofpetrolatum and a fatty alcohol such as cetyl or stearyl alcohol; theaqueous phase usually, although not necessarily, exceeds the oil phasein volume, and generally contains a humectant. The emulsifier in a creamformulation is generally a nonionic, anionic, cationic or amphotericsurfactant. The specific ointment or cream base to be used, as will beappreciated by those skilled in the art, is one that will provide foroptimum drug delivery. As with other carriers or vehicles, an ointmentbase should be inert, stable, nonirritating and nonsensitizing.

IV. Mitigation/Treatment of Cancers.

In certain instances the p53 mimetics/agonists of this invention areadministered to mitigate one or more symptoms of a cancer (e.g., toinduce cell death in cancer cells). The p53 mimetics/agonists can beadministered to reduce tumor growth/proliferation, to inhibitmetastasis, to prevent relapse (e.g. after surgery, radiotherapy, etc.),or as a component of a multiple-modality therapy.

Typically the p53 mimetics/agonists will be administered in an amountsufficient to produce a biological effect (e.g., to inhibit cancer cellgrowth and/or proliferation). Greatest effeciacy is expected to be foundin cancers characterized by p53 naïve cells.

V. Kits.

In still another embodiment, this invention provides kits for screeningfor p53 mimetics/agonists. Such kits typically comprise a containercontaining a cell line as described herein.

In other embodiments, this invention provides kits promoting cell deathin a p53 naïve cell. These kits typically comprise a containercontaining one or more of the p53 mimetics/agonists of this invention.

The kits may optionally include one or more reagents for use in themethods of this invention. Such “reagents” may include, but are notlimited to, cells and/or cell lines, transfection reagents (e.g. CaPO₄,lipofectin), detectable labels, means for detecting labels, buffers,anti-transporter antibodies, nucleic acid constructs encodinghousekeeping genes, bioreactors, syringes, and other devices.

In addition, the kits may include instructional materials containingdirections (i.e., protocols) for the practice of the methods of thisinvention. Certain preferred instructional materials provide protocolsutilizing the kit contents for screening for p53 mimetics/agonists orfor promoting cell death in p53 naïve cells. While the instructionalmaterials typically comprise written or printed materials they are notlimited to such. Any medium capable of storing such instructions andcommunicating them to an end user is contemplated by this invention.Such media include, but are not limited to electronic storage media(e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g.,CD ROM), and the like. Such media may include addresses to internetsites that provide such instructional materials.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1 Identification of P53 Mimetics

To identify p53 mimics, we developed a live mammalian cell system thatreports the promotion of the expression of the pro-apoptotic Baxprotein. The bax promoter consists of a 372 nucleotide 5′ UTR, a TATAAsequence, and several consensus sequences that bind p53, resulting inup-regulation of Bax expression (see, e.g., FIG. 1). By expressing thesesites upstream of the gene encoding luciferase in HEK293T cells, we haveestablished a novel set of reporter cell lines. These cells provide asystem which allows identification of both direct activators of Baxexpression and replacements of an apoptotic function of p53.

Discovery of a Small Molecule that Replaces p53 Function

In initial studies, cotransfection of a p53 expression plasmid with areporter bearing oligomerized consensus binding sites yielded stronglyluminescent cells, whereas cotransfection with an irrelevant plasmidgave no luminescence. Our system shows high enough signal and low enoughbackground to permit small molecules to be isolated from compoundlibraries. By employing the Multi-Probe H liquid handling robotic systemand the ultra-sensitive Top-Count luminometer, high-speed screening ofthousands of compounds has been enabled at the Buck Institute.

We obtained and screened a 2000 compound small molecule library madeavailable by the National Cancer Institute in our p53 reporter system inHEK293T cells. Surprisingly, we identified eight compounds that actedupon the p53 reporter system and yielded increases in luciferaseexpression and activity. One compound (dubbed Rp53-1 or G6) producedsignificant levels of luciferase activity, and at high concentrations(10 μM) induced expression of luciferase levels greater than transfectedwild-type p53 (see, e.g., FIG. 2B).

Chemotherapeutic Agent Rp53-1 Specifically Targets Neoplasia

To assess the ability of the small molecule compound Rp53-1 to mimic thetranscription-promoting functions of p53 leading to Bax expression andinduction of cell death, p53-mutated MDA-MB-435 and p53-wild-type MCF-7human breast cancer cells were treated with Rp53-1. Interestingly onlythe former was sensitive to Rp53-1 (FIG. 3). Similarly, other p53-mutanttumor cell lines were also sensitive to Rp53-1 (colon: ColoHSR, SW480,HT29; prostate: PC-3, DU145; osteosarcoma: SAOS-2), whereasp53-wild-type cells were resistant (colon: Lovo; prostate: LNCaP) Thespecificity displayed by Rp53-1 in inducing death solely in p53-mutantcells makes it an ideal candidate for utilization as a therapeuticintervention for p53-inactive (or p53-malfunctioning) tumors (i.e., mostclinically relevant tumors). Lack of toxicity in cells generating normalp53 activity (all other human cells) would reduce possible side effects.

Since p53 upregulates Bax expression while it also downregulates therelated anti-apoptotic Bcl-2 (thus having at least two effects that leadto cell death), the identification of small molecules able to replacelost p53 activity by upregulating Bax, thus inducing cell death in ap53-independent fashion (i.e., inducing cell death in the absence of p53function by replacing p53 functional activity with a small molecule) mayallow the discovery of novel tools to control both Bax and Bcl-2 withintissues. The importance of these critical molecules in human neoplasiais supported by the high frequency of p53, Bax and Bcl-2 mutations inhuman cancers.

CONCLUSION

The discovery of the first p53 mimic, the active compound Rp53-1,validates and displays the potential of the methodology outlined above.The screening systems described herein significantly increase ourpotential to make discoveries like Rp53-1, optimize Rp53-1 for humanclinical trials, and make possible a novel approach to eradicate breastcancer.

Example 2 Cancer Therapeutics Using p53 Mimetics as Activators of BaxExpression

p53 mutations are associated with a high percentage of all human tumors.Using a new high-throughput small molecule screen developed here,scientists in the Buck Institute's Discovery Translation Unit (DTU) havediscovered the first p53 functional mimetic called G6. G6 and relatedtherapeutics can be developed as therapeutic agents for cancersincluding breast, prostate, lung, colon and pancreatic cancers as wellas osteosarcoma.

BACKGROUND

p53 is regarded to be the “guardian of the genome.” One of p53's rolesis to commit genetically abnormal cells to programmed cell death therebysuppressing tumor growth. If p53 is absent or mutated to anon-functional form its key role as a tumor suppressor is eliminated, asis observed in many cancers. Activation of p53 is often necessary forsensitizing tumor cells to chemotherapy and radiation; absence of p53function is often associated with unresponsiveness to these therapies.Over one-half of all human tumors lack proper p53 function. Notsurprisingly, methods of restoring p53 function have long been sought asan anticancer strategy, however, this has proven to be a dauntingproblem.

One key function for p53 is to regulate the expression of proteinsimportant to the life and death decisions that cells make whenresponding to adverse, neoplastic situations, such as deleteriousmutations introduced in DNA or unwarranted increases in rate ofproliferation. Activation of p53 (by modifications such asphosphorylation (FIG. 4, circle) results in alterations in theexpression of such proteins as the antagonistic partners Bax and Bcl2.

In neoplastic conditions, p53 upregulates the expression of thepro-death protein Bax, while it downregulates the anti-death (oncogenic)protein Bcl2. Moreover, the only known endogenous activator of Baxexpression is p53. One-third of all advanced human breast carcinomasdemonstrate a marked reduction in expression of Bax. The subgroup ofpatients displaying reduced Bax expression generally respond poorly totherapy and exhibit rapidly growing tumors and shorter median survival.For these and other reasons, we have designed a screen for smallmolecule compounds that mimic the role of p53 as a retardant ofoncogenesis.

THE TECHNOLOGY

Screening for small molecule compounds that act as p53 mimics isexpected to yield therapeutics for many cancers. The discovery of thefirst p53 functional mimic, the active compound G6 (see, e.g., FIGS. 2Aand 2B) validates and displays the potential of the methodology.Utilizing a novel live-cell high-throughput screening system wedetermined that G6 (FIG. 2A) remediates the transcriptional activitiesof p53 lost in neoplasia. This “lead” compound presents the opportunityfor optimization for human clinical trial in cancers which are p53deficient or which express faulty p53. Clearly other compound familiescan be identified through the same screening process.

Inherent in the initial drug design was a strategy to develop a therapythat targets mutant p53 containing cells and that would leave healthycells expressing normal p53 unaffected (FIG. 3). Affirming the validityof G6 as a lead compound, every p53 defective tumor cell line treatedwith G6 showed susceptibility, whereas wild type cells fromcorresponding normal tissue were unaffected thus confirming the drug'sselectivity (see, e.g., Table 1, and FIGS. 8 and 9).

TABLE 1 P53 wt and null cells are sensitive to G6. P53 Status Mutant orG6 Cell Line WT Null Sensitivity Prostate PC3 Null ++ DU145 Mt ++ LNcapWT − Colon ColoHSR Mt ++++ SW480 Mt ++ Lovo WT − HT29 Mt +++ BreastMDA-435 Mt +++ MCF-7 WT − Osteosarcoma Saos-2 Null ++++ Lung A549 WT −H23 Mt +++ Embryonic kidney 293 WT −

Since G6 activates an inherent cellular pathway, a single pulse of G6 inthe nanomolar range is sufficient to activate the cell death program(FIG. 7). Thus G6 appears to be a potent treatment.

Initial trials of G6 in human osteosarcoma xenografted nude mice haveshown promise (FIG. 10). Mice bearing p53 abnormal tumors have respondedwell to G6. Tumor regression has been complete and none of the mice haveshown adverse side effects in response to treatment with G6. Furthermouse studies are currently underway.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

1-13. (canceled)
 14. A cell line for screening for agents that promotescell death in a p53 naive cell, said cell line comprising mammaliancells containing a nucleic acid construct comprising bax promoterelements comprising a 5′ UTR, a TATAA sequence, and one or moreconsensus sequences that bind p53, where said bax promoter elements areoperably linked to a reporter such that binding of a p53 protein to oneor more of said consensus sequences induces or increases transcriptionof said reporter.
 15. The cell line of claim 14, wherein said reporteris a reporter selected from the group consisting of fluorescent protein,luciferase, chloramphenicol acetyl transferase (CAT), β-galactosidase(β-gal), alkaline phosphatase, horse radish peroxidase (HRP), and growthhormone (GH).
 16. The cell line of claim 14, wherein said reporter is aluciferin gene.
 17. The cell line of claim 14, wherein said mammaliancell is a p53 naïve cell.
 18. The cell line of claim 14, wherein saidmammalian cell is an HEK293T cell.
 19. A method of identifying agentsthat promote cell death in p53 naive cells, said method comprising:contacting a cell from a cell line according to claim 14 with one ormore test agents; and detecting a signal from the reporter in said cellwherein an increase in signal from said reporter indicates that saidagent is an agent that is likely to promote cell death in a p53 naivecell.
 20. The method of claim 19, where a plurality of test agents arecontacted to said cell at the same time.
 21. The method of claim 19,where a single test agent is contacted to said cell.
 22. The method ofclaim 19, wherein said increase is relative to a control lacking saidtest agent.
 23. The method of claim 19, wherein said increase isrelative to a control comprising said test agent at a lowerconcentration.
 24. The method of claim 19, wherein said method isperformed by a robotic system.
 25. The method of claim 19, wherein saidtest agents are from a library selected from the group consisting ofChem Bridge DiverSet E, Bionet 1, CEREP, Maybridge 1, Maybridge 2,Peakdale 1, Peakdale 2, ChemDiv Combilab and International, MixedCommercial Plate 1, Mixed Commercial Plate 2, Mixed Commercial Plate 3,Mixed Commercial Plate 4, ChemBridge Microformat, Commercial DiversitySet1, NCI Structural Diversity Set version 1, NCI Structural DiversitySet version 2, NCI Mechanistic Diversity Set, NCI Open Collection 1, NCIOpen Collection 2, NINDS Custom Collection, SpecPlus Collection, BIOMOLICCB Known Bioactives, ICCB Discretes Collections, ICCB2, ICCB3, ICCB4,NCI Marine Extracts, Aqueous fractions—NCI Plant and Fungal Extracts,Organic fractions—NCI Plant and Fungal Extracts, Philippines PlantExtracts 1, Philippines Plant Extracts 2, and Starr Foundation Extracts1.